The synthesis of nucleotides in organisms is required for the cells in those organisms to divide and replicate. Nucleotide synthesis in mammals may be achieved through one of two pathways: the de novo synthesis pathway or the salvage pathway. Different cell types use these pathways to a different extent.
Inosine-5'-monophosphate dehydrogenase (IMPDH; EC 1.1.1.205) is an enzyme involved in the de novo synthesis of guanosine nucleotides. IMPDH catalyzes the NAD-dependent oxidation of inosine-5'-monophosphate (IMP) to xanthosine-5'-monophosphate (XMP), which is the committed step in guanosine nucleotide synthesis. [R. C. Jackson et. al., Nature, 256, pp. 331-333 (1975)].
IMPDH is ubiquitous in eukaryotes, bacteria and protozoa (Y. Natsumeda and S. F. Carr, Ann. N.Y. Acad. Sci., 696, pp. 88-93 (1993). The prokaryotic forms share 30-40% sequence identity with the human enzyme. Regardless of species, the enzyme follows an ordered Bi--Bi reaction sequence, where IMP binding precedes that of NAD, and NADH is released prior to XMP [S. F. Carr et al., J. Biol. Chem. 268, pp. 27286-27290 (1993); E. W. Holmes; Biochim. Biophys. Acta 364, pp. 209-217 (1974)]. This mechanism differs from that of most other known NAD-dependent dehydrogenases, which have either a random order of substrate addition or require that NAD bind before substrate.
The de novo synthesis of guanosine nucleotides, and thus the activity of IMPDH, is particularly important in B- and T-lymphocytes. These cells depend on the de novo, rather than salvage pathway to generate sufficient levels of nucleotides necessary to initiate a proliferative response to mitogen or antigen [A. C. Allison et. al., Lancet II, 1179 (1975) and A. C. Allison et. al., Ciba Found. Symp., 48, 207 (1977)]. Thus, IMPDH is an attractive target for selectively inhibiting the immune system without also inhibiting the proliferation of other cells.
Immunosuppression has been achieved by inhibiting a variety of enzymes including for example, the phosphatase calcineurin (inhibited by cyclosporin A and FK-506); dihydroorotate dehydrogenase, an enzyme involved in the biosynthesis of pyrimidines (inhibited by leflunomide and brequinar); the kinase FRAP (inhibited by rapamycin); and the heat shock protein hsp70 (inhibited by deoxyspergualin). [See B. D. Kahan, Immunological Reviews, 136, pp. 29-49 (1993); R. E. Morris, The Journal of Heart and Lung Transplantation, 12(6), pp. S275--S286 (1993)].
Inhibitors of IMPDH are also known. U.S. Pat. Nos. 5,380,879 and 5,444,072 and PCT publications WO 94/01105 and WO 94/12184 describe mycophenolic acid (MPA) and some of its derivatives as potent, uncompetitive, reversible inhibitors of human IMPDH type I and type II. MPA has been demonstrated to block the response of B and T-cells to mitogen or antigen [A. C. Allison et. al., Ann. N. Y. Acad. Sci., 696, 63 (1993). ##STR1##
Immunosuppressants, such as MPA, are useful drugs in the treatment of transplant rejection and autoimmune diseases [R. E. Morris, Kidney Intel., 49, Suppl. 53, S-26 (1996)]. MPA, however, is characterized by undesirable pharmacological properties, such as gastrointestinal toxicity and poor bioavailability. [L. M. Shaw, et. al., Therapeutic Drug Monitoring, 17, pp. 690-699, (1995)].
Nucleoside analogs such as tiazofurin, ribavirin and mizoribine also inhibit IMPDH [L. Hedstrom et. al., Biochemistry, 29, pp. 849-854 (1990)]. These compounds, however, are not specific for IMPDH.
Mycophenolate mofetil, a prodrug which quickly liberates free MPA in vivo, was recently approved to prevent acute renal allograft rejection following kidney transplantation [L. M. Shaw et al., Therapeutic Drug Monitoring, 17, pp. 690-699 (1995); H. W. Sollinger, Transplantation, 60, pp. 225-232 (1995)]. However, because of gastrointestinal and other side-effects, the therapeutic potential of this drug appears limited [L. M. Shaw et al., Therapeutic Drug Monitoring, 17, pp. 690-699 (1995); A. C. Allison and E. M. Eugui Immunological Rev., 136, pp. 5-28 (1993)].
It is also known that IMPDH plays a role in other metabolic events. Increased IMPDH activity has been observed in rapidly proliferating human leukemic cell lines and other tumor cell lines, indicating IMPDH is a target for anti-cancer as well as immunosuppressive chemotherapy [M. Nagai et. al., Cancer Res., 51, pp. 3886-3890 (1991)].
IMPDH has also been shown to play a role in the proliferation of smooth muscle cells, indicating that inhibitors of IMPDH, such as MPA or rapamycin, may be useful in preventing restenosis or other hyperproliferative vascular diseases [C. R. Gregory et al., Transplantation, 59, pp. 655-61 (1995); PCT publication WO 94/12184; and PCT publication WO 94/01105].
Additionally, IMPDH has been shown to play a role in viral replication in some viral cell lines (S. F. Carr, J. Biol. Chem., 268, pp. 27286-27290 (1993)]. Analogous to lymphocyte and tumor cell lines, the implication is that the de novo, rather than the salvage, pathway is critical in the viral replication process.
Thus, there remains a need for potent IMPDH inhibitors with improved pharmacological properties. Such inhibitors would have therapeutic potential as immunosuppressants, anti-cancer agents, anti-vascular hyperproliferative agents and anti-viral agents. Specifically, such compounds may be used in the treatment of transplant rejection and autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, juvenile diabetes, asthma, inflammatory bowel disease, as well as in the treatment of cancer and tumors, such as lymphomas and leukemia, vascular diseases, such as restenosis, and viral replication diseases, such as retroviral diseases and herpes.
Two isoforms of human IMPDH, designated type I and type II, have been identified [F. R. Collart and E. Huberman, J. Biol. Chem., 263, pp. 15769-15772 (1988); Y. Natsumeda et. al., J. Biol. Chem., 265, pp. 5292-5295 (1990)]. Each is 514 amino acids, and they share 84% sequence identity. Both IMPDH type I and type II form active tetramers in solution, with subunit molecular weights of 56 kDa [Y. Yamada et. al., Biochemistry, 27, pp. 2737-2745 (1988)].
Both human IMPDH isoforms have been characterized by their cDNA and amino acid sequences [Y. Natsumeda et. al., J. Biol. Chem., 265, pp. 5222-5295 (1990)]. Chinese hamster IMPDH has been characterized by its cDNA and its amino acid sequence [F. R Collart and E. Hubermann, J. Biol. Chem., 263, pp. 15769-15772 (1988). Knowledge of the primary structure, i.e., amino acid sequence, of IMPDH, however, does not allow prediction of its tertiary structure. Nor does it afford an understanding of the structural, conformational and chemical interactions of IMPDH with MPA, IMP, or other compounds or inhibitors.
The crystal structure of IMPDH has not been reported. Nor has the crystal structure of a IMPDH homologue or a IMPDH co-complex been reported. The need, therefore, exists for determining the crystal structure of IMPDH to provide a more accurate description of the structure of IMPDH to aid in the design of improved IMPDH inhibitors. The crystal structure of a complex comprising IMPDH, IMP and MPA would provide such a description.